The Diving Response Mechanism and its Surprising Evolutionary Path in Seals andSea Lions

During the last half century or more, studies of diving physiologyand biochemistry made great progress in mechanistically explainingthe basic diving response of aquatic mammals and birds. Keycomponents of the diving response (apnea, bradycardia, peripheralvasoconstriction, redistribution of cardiac output) were foundin essentially all species analyzed and were generally takento be biological adaptations. By the mid 1970s, this approachto unravelling the diving response had run 'out of steam' andwas in conceptual stasis. The breakthrough which gave renewalto the field at this time was the development of microprocessorbased monitoring of diving animals in their natural environments,which led to a flurry of studies mostly confirming the basicoutlines of the diving response based upon laboratory studiesand firmly placing it into proper biological context, underliningits plasticity and species specificities. Now towards the endof the millenium, despite ever more detailed field monitoringof physiology, behaviour and ecology, mechanistic studies areagain approaching a point of diminishing returns. To avoid anotherconceptual stasis, what seems required are new initiatives whichwe anticipate may arise from two differing approaches. The firstis purely experimental, relying on magnetic resonance imaging(MRI) and spectroscopy (MRS) to expand the framework of theoriginal "diving response" concept. The second—evolutionarystudy of the diving response—is synthetic, linked to bothfield and laboratory studies. To date the evolution of the divingresponse has only been analyzed in pinnipeds and from thesestudies two kinds of patterns have emerged. (1) Some physiologicaland biochemical characters, required and used in diving animals,are highly conserved not only in pinnipeds but in all vertebrates;these traits are necessarily similar in all pinnipeds and includediving apnea, bradycardia, tissue specific hypoperfusion, andhypometabolism of hypoperfused tissues. (2) Another group offunctionally linked characters are more malleable and include(i) spleen mass, (ii) blood volume, and (iii) hemoglobin (Hb)pool size. Increases in any of these traits improve diving capacity.Assuming that conserved physiological function means conservedsequences in specific genes and their products (and that evolvingfunction requires changes in such sequences), it is possibleto rationalize both above trait categories in pinniped phytogeny.However, it is more difficult for molecular evolution theoryto explain how complex regulatory systems like those involvedin bradycardia and peripheral vasoconstriction remain the samethrough phylogenetic time than it is to explain physiologicalchange driven by positive natural selection.     

Defining the limits of diving biochemistry in marine mammals

The field of marine mammal diving biochemistry was essentially untouched when Peter Hochachka turned his attention to it in the mid-1970s. Over the next 30 years, his work followed three main themes in this area: first, most biologists at that time supported the theory that diving mammals utilized enhanced metabolic pathways for hypoxic energy production (glycolysis to lactate) and reduced their metabolic rate while diving. Peter began his work on potential hypoxic adaptations in marine mammals by working out the details of how these pathways would be regulated. By the 1980s, he started to ask how diving mammals balanced the increased demands of exercise with the apparently conflicting demands to reduce aerobic metabolism while exercising underwater. By the 1990s, his work involved complex models of the interplay between the neural, hormonal, behavioral and evolutionary components of diving biochemistry and animal exercise. From a comparative approach, he excelled at bringing themes of hypoxic adaptation from many different types of animals to the field of diving mammal biochemistry. This review traces the history of Peter Hochachka's work on diving biochemistry from the perspective of those of us who spent time with him both inside the laboratory and outside in the field from Antarctica to Iceland.     

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.     

The history and purview of phylogeography: a personal reflection

Last year marked the 10th anniversary of the birth of phylogeography as a formal discipline. However, the field's gestation began in the mid-1970s with the introduction of mitochondrial (mt) DNA analyses to population genetics, and to the profound shift toward genealogical thought at the intraspecific level (now formalized as coalescent theory) that these methods prompted. This paper traces the early history and explosive growth of phylogeography, and closes with predictions about future challenges for the field that centre on several facets of genealogical concordance.     

Repetitive paired stimulation of nasotrigeminal and peripheral chemoreceptor afferents cause progressive potentiation of the diving bradycardia

Hallmarks of the mammalian diving response are protective apnea and bradycardia. These cardiorespiratory adaptations can be mimicked by stimulation of the trigeminal ethmoidal nerve (EN5) and reflect oxygen-conserving mechanisms during breath-hold dives. Increasing drive from peripheral chemoreceptors during sustained dives was reported to enhance the diving bradycardia. The underlying neuronal mechanisms, however, are unknown. In the present study, expression and plasticity of EN5-bradycardias after paired stimulation of the EN5 and peripheral chemoreceptors was investigated in the in situ working heart-brain stem preparation. Paired stimulations enhanced significantly the bradycardic responses compared with EN5-evoked bradycardia using submaximal stimulation intensity. Alternating stimulations of the EN5 followed by paired stimulation of the EN5 and chemoreceptors (10 trials, 3-min interval) caused a progressive and significant potentiation of EN5-evoked diving bradycardia. In contrast, bradycardias during paired stimulation remained unchanged during repetitive stimulation. The progressive potentiation of EN5-bradycardias was significantly enhanced after microinjection of the 5-HT(3) receptor agonist (CPBG hydrochloride) into the nucleus tractus solitarii (NTS), while the 5-HT(3) receptor antagonist (zacopride hydrochloride) attenuated the progressive potentiation. These results suggest an integrative function of the NTS for the multimodal mediation of the diving response. The potentiation or training of a submaximal diving bradycardia requires peripheral chemoreceptor drive and involves neurotransmission via 5-HT(3) receptor within the NTS.     

Simulated human diving and heart rate: making the most of the diving response as a laboratory exercise

Laboratory exercises in which students examine the human diving response are widely used in high school and college biology courses despite the experience of some instructors that the response is unreliably produced in the classroom. Our experience with this exercise demonstrates that the bradycardia associated with the diving response is a robust effect that can easily be measured by students without any sophisticated measurement technology. We discuss measures that maximize the success of the exercise by reducing individual variation, designing experiments that are minimally affected by change in the response over time, collecting data in appropriate time increments, and applying the most powerful statistical analysis. Emphasis is placed on pedagogical opportunities for using this exercise to teach general principles of physiology, experimental design, and data analysis. Data collected by students, background information for instructors, a discussion of the relevance of the diving reflex to humans, suggestions for additional experiments, and thought questions with sample answers are included.     

The effects of breathing different levels of O2 and CO2 on the diving responses of ducks (Anas platyrhynchos) and cormorants (Phalacrocorax auritus)

Summary The effects of breathing different levels of O₂ and CO₂ before forced dives were investigated in 5 dabbling ducks (White Pekin) and 5 deep divers (Double Crested Cormorants). Breathing and heart rates, blood gases, and blood pH, were monitored. After breathing air before diving, ducks exhibited a slow decrease in heart rate that reached a minimum of 20 beats·min⁻¹ after 50 s submergence. The development of bradycardia was retarded if the duck breathed a hyperoxic gas mixture before diving and was accelerated if the gas mixture was hypoxic and hypercapnic. The cormorants' diving heart rate decreased to a minimum of about 60 beats·min⁻¹ in less than 20 s and development of bradycardia was unaffected by different levels of O₂ and CO₂ breathed before diving. Consequently, bradycardia in forced dived cormorants was unrelated to changes in blood gases in the dives which suggests that intravascular chemoreceptors are unimportant in initiating diving bradycardia in cormorants.     

Deadly diving? Physiological and behavioural management of decompression stress in diving mammals

Decompression sickness (DCS; 'the bends') is a disease associated with gas uptake at pressure. The basic pathology and cause are relatively well known to human divers. Breath-hold diving marine mammals were thought to be relatively immune to DCS owing to multiple anatomical, physiological and behavioural adaptations that reduce nitrogen gas (N(2)) loading during dives. However, recent observations have shown that gas bubbles may form and tissue injury may occur in marine mammals under certain circumstances. Gas kinetic models based on measured time-depth profiles further suggest the potential occurrence of high blood and tissue N(2) tensions. We review evidence for gas-bubble incidence in marine mammal tissues and discuss the theory behind gas loading and bubble formation. We suggest that diving mammals vary their physiological responses according to multiple stressors, and that the perspective on marine mammal diving physiology should change from simply minimizing N(2) loading to management of the N(2) load. This suggests several avenues for further study, ranging from the effects of gas bubbles at molecular, cellular and organ function levels, to comparative studies relating the presence/absence of gas bubbles to diving behaviour. Technological advances in imaging and remote instrumentation are likely to advance this field in coming years.     

Diving physiology of birds: a history of studies on polar species

Our knowledge of avian diving physiology has been based primarily on research with polar species. Since Scholander's 1940 monograph, research has expanded from examination of the 'diving reflex' to studies of free-diving birds, and has included laboratory investigations of oxygen stores, muscle adaptations, pressure effects, and cardiovascular/metabolic responses to swimming exercise. Behavioral and energetic studies at sea have shown that common diving durations of many avian species exceed the calculated aerobic diving limits (ADL). Current physiological research is focused on factors, such as heart rate and temperature, which potentially affect the diving metabolic rate and duration of aerobic diving.     

Laurence Irving: an appreciation

Laurence Irving (1895-1979) contributed significantly over five decades to the development of environmentally oriented physiological studies. He is best known for his investigations of the physiology of diving mammals, the respiratory properties of fish blood, and cold adaptation and acclimatization in poikilotherms and homeotherms, including man. Beyond his own research contributions, Irving benefited American comparative physiology through his key roles in the immigration of Per F. Scholander and Knut and Bodil Schmidt-Nielsen to the United States. The Irving-Scholander research collaboration provides a substantial legacy for comparative physiology. Laurence Irving's administrative contributions include service as the first scientific director of the Arctic Research Laboratory at Barrow, Alaska, and as the founding director of the Institute of Arctic Biology at the University of Alaska, Fairbanks. These units have assured the implementation of his philosophy of combining laboratory and field studies in the investigation of environmentally oriented physiological problems. Laurence Irving was an ardent advocate for Alaskan research, and his efforts were an important help in the advancement of science in the state.     

Diving behaviour of a reptile (Crocodylus johnstoni) in the wild: interactions with heart rate and body temperature

The differences in physical properties of air and water pose unique behavioural and physiological demands on semiaquatic animals. The aim of this study was to describe the diving behaviour of the freshwater crocodile Crocodylus johnstoni in the wild and to assess the relationships between diving, body temperature, and heart rate. Time-depth recorders, temperature-sensitive radio transmitters, and heart rate transmitters were deployed on each of six C. johnstoni (4.0-26.5 kg), and data were obtained from five animals. Crocodiles showed the greatest diving activity in the morning (0600-1200 hours) and were least active at night, remaining at the water surface. Surprisingly, activity pattern was asynchronous with thermoregulation, and activity was correlated to light rather than to body temperature. Nonetheless, crocodiles thermoregulated and showed a typical heart rate hysteresis pattern (heart rate during heating greater than heart rate during cooling) in response to heating and cooling. Additionally, dive length decreased with increasing body temperature. Maximum diving length was 119.6 min, but the greatest proportion of diving time was spent on relatively short (<45 min) and shallow (<0.4 m) dives. A bradycardia was observed during diving, although heart rate during submergence was only 12% lower than when animals were at the surface.     

Characteristics of the human cardiovascular system in the human diving response

Comparative-evolutional research of diving response showed that mechanisms of its expression had much in common in humans and in animals. Firstly, it involves a reflex bradycardia, vasoconstriction of peripheral vessels, and blood flow centralization. But, unlike animals whose diving response has some typical species peculiarities, human diving response is rather diverse. Four types of cardiovascular system response to face submersion were revealed: over-reactive, reactive, paradoxical, and nonreactive. These types were chosen according to the bradycardia character. It is also supposed that the occurrence of individual maximal R--R-interval, while serving as a signal to apnea stopping, is among the reasons of apnea activity limitation.     

The human spleen as an erythrocyte reservoir in diving-related interventions.

Twelve subjects without and ten subjects with diving experience performed short diving-related interventions. After labeling of erythrocytes, scintigraphic measurements were continuously performed during these interventions. All interventions elicited a graduated and reproducible splenic contraction, depending on the type, severity, and duration of the interventions. The splenic contraction varied between approximately 10% for "apnea" (breath holding for 30 s) and "cold clothes" (cold and wet clothes applied on the face with no breath holding for 30 s) and approximately 30-40% for "simulated diving" (simulated breath-hold diving for 30 s), "maximal apnea" (breath holding for maximal duration), and "maximal simulated diving" (simulated breath-hold diving for maximal duration). The strongest interventions (simulated diving, maximal apnea, and maximal simulated diving) elicited modest but significant increases in hemoglobin concentration (0.1-0.3 mmol/l) and hematocrit (0.3-1%). By an indirect method, the splenic venous hematocrit was calculated to 79%. No major differences were observed between the two groups. The splenic contraction should, therefore, be included in the diving response on equal terms with bradycardia, decreased peripheral blood flow, and increased blood pressure.     

Diving heart rate development in postnatal harbour seals, Phoca vitulina

Harbour seals, Phoca vitulina, dive from birth, providing a means of mapping the development of the diving response, and so our objective was to investigate the postpartum development of diving bradycardia. The study was conducted May-July 2000 and 2001 in the St. Lawrence River Estuary (48 degrees 41'N, 68 degrees 01'W). Both depth and heart rate (HR) were remotely recorded during 86,931 dives (ages 2-42 d, n = 15) and only depth for an additional 20,300 dives (combined data covered newborn to 60 d, n = 20). The mean dive depth and mean dive durations were conservative during nursing (2.1 +/- 0.1 m and 0.57 +/- 0.01 min, range = 0-30.9 m and 0-5.9 min, respectively). The HR of neonatal pups during submersion was bimodal, but as days passed, the milder of the two diving HRs disappeared from their diving HR record. By 15 d of age, most of the dive time was spent at the lower diving bradycardia rate. Additionally, this study shows that pups are born with the ability to maintain the lower, more fully developed dive bradycardia during focused diving but do not do so during shorter routine dives.     

CHANGES IN HEART RATE VARIABILITY DURING DIVING IN YOUNG HARBOR SEALS, PHOCA VITULINA

We studied changes in the patterns of heart rate variability (HRV) that coincide with the development of diving skills in harbor seal pups, Phoca vitulina . Heart rate measurements were collected remotely. Spectral analysis of HRV revealed power within a mid-frequency band (0.1–0.3 Hz) which was prominent, especially in pups less than 10 d of age. In these younger pups, the heart rate switched cyclically between a low and a high diving heart rate every 3–10 sec. Older pups exhibited a highly controlled diving bradycardia with a lower median and a lower variance when compared to younger individuals. These results provide new insight into the maturation of the bradycardia component of the dive response in harbor seal pups.     

Balancing conflicting metabolic demands of exercise and diving

During enforced diving, aquatic animals activate a set of physiological reflexes (apnea, bradycardia, peripheral vasoconstriction), which are termed the diving response and are in effect the first line of defense against hypoxia. At least in the Weddell seal, this strategy is now known also to be used in voluntary diving at sea, but the response is necessarily modified to accommodate potentially conflicting demands of diving and swimming exercise. The main modification appears to involve skeletal muscles used in swimming, which, because of their high energy requirements, must be powered by aerobic metabolism. Thus they must remain perfused at rates porportional to swimming velocity (which is why heart rates are adjusted to swimming velocity). The required regulation of O2 delivery is achieved at least in part by a well-paced release of oxygenated red blood cells, stored at the beginning of the dive apparently in the spleen. The main metabolic difference between laboratory and voluntary diving is that, in the latter, working muscles serve as a sink for lactate and thus the entry rates of lactate into the plasma can be balanced by exit rates from the plasma; the maintenance of this balance means that no excess lactate remains for a lactate washout in postdiving exercise except under long, exploratory diving. Even in the latter long dives, however, the amount of lactate formed is far less than would be expected if the energetic shortfall caused by hypoperfusion and O2 lack were made up by anaerobic glycolysis (Pasteur effect). Consequently, during diving, hypoperfused tissues necessarily sustain a metabolic arrest of variable degrees as a mechanism of defense against hypoxia.     

Trial Implantation of Heart Rate Data Loggers in Pinnipeds

ABSTRACT Pinnipeds are major consumers in marine ecosystems, and understanding their energy budgets is essential to determining their role in food webs, particularly where there is competition with fisheries. Food consumption and energy expenditure have been evaluated in pinnipeds using different methods, but the use of heart rate to estimate energy expenditure is potentially a very powerful tool suited to the life history of these animals. We tested a procedure for the subcutaneous implantation of heart rate data loggers to determine whether heart rate could be recorded for ≥1 year in free-ranging pinnipeds, as it has been in birds. We implanted 3 captive California sea lions (Zalophus californianus) and 3 captive northern elephant seals (Mirounga angustirostris) with heart rate data loggers and monitored their recovery and behavior in a controlled environment. In both species, the implantation site allowed for excellent detection of the electrocardiogram, and we observed heart rate signatures characteristic of behaviors such as resting and diving. Although all 3 sea lions recovered well from the implantation surgery, all 3 elephant seals showed a substantial inflammatory response for unknown reasons, and we removed the implanted data loggers. Subcutaneous implantation of data loggers is a powerful technique to study physiology, energetics, and behavior in California sea lions, but more work is required to realize the potential of this technique in northern elephant seals.     

The development of the pulmonary surfactant system in California sea lions

Pulmonary surfactant has previously been shown to change during development, both in composition and function. Adult pinnipeds, unlike adult terrestrial mammals, have an altered lung physiology to cope with the high pressures associated with deep diving. Here, we investigated how surfactant composition and function develop in California sea lions (Zalophus californianus). Phosphatidylinositol was the major anionic phospholipid in the newborn, whereas phosphatidylglycerol was increased in the adult. This increase in phosphatidylglycerol occurred at the expense of phosphatidylinositol and phosphatidylserine. There was a shift from long chain and polyunsaturated phospholipid molecular species in the newborn to shorter chain and mono- and disaturated molecular species in the adult. Cholesterol and SP-B concentrations were also higher in the adult. Adult surfactant could reach a lower equilibrium surface tension, but newborn surfactant could reach a lower minimum surface tension. The composition and function of surfactant from newborn California sea lions suggest that this age group is similar to terrestrial newborn mammals, whereas the adult has a "diving mammal" surfactant that can aid the lung during deep dives. The onset of diving is probably a trigger for surfactant development in these animals.     

The human heart rate response profiles to five vagal maneuvers

Healthy teens and adults performed four vagotonic maneuvers. A large series of strabismus surgery patients had deliberately quantified tension on extraocular rectus muscles during general anesthesia. The mean bradycardia was greatest for diving response (apneic facial exposure to cold) and Valsalva maneuver and least for pressure on the globe and carotid sinus massage. Bradycardia occurred for every subject for the non-surgical maneuvers, however, extraocular muscle tension frequently caused no change in heart rate or even tachycardia. The inter-subject variance in percent heart rate change was greatest for surgical oculocardiac reflex. Of the rectus muscles, the inferior caused the most bradycardia while the lateral caused the least. The percent oculocardiac reflex was not age dependent. Occasional patients demonstrated profound bradycardia with strabismus surgery. Of these maneuvers, diving response has theoretical advantage in treating paroxysmal atrial tachycardia. The human cardiac vagal efferent was stimulated by several carefully controlled maneuvers resulting in wide inter-maneuver differences in bradycardia magnitude. The greatest intra-maneuver variability occurred with surgical oculocardiac reflex.     

DARK ADAPTATION AND VISUAL SENSITIVITY IN SHALLOW AND DEEP-DIVING PINNIPEDS1

Pinnipeds forage almost exclusively underwater. Consequently, observing them is difficult and relatively little is known of how they use their senses to locate prey, avoid predators, and navigate while diving. Vision has been presumed to be of primary importance, although previous measurements of visual functioning in pinnipeds have been restricted to just a few shallow-diving species. As diving pinnipeds experience rapid changes in light levels during descent/ascent and low light levels at depth, it has not been clear whether they possess visual capabilities adequate for use while diving, particularly in the case of deep-diving species. To examine this issue, behavioral psychophysics have been used to assess and compare the dark adaptation rates and relative light sensitivities of a deep-diving pinniped (northern elephant seal, Mirounga angustirostris), two shallow-diving species (California sea lion, Zalophus californianus, and harbor seal, Phoca vitulina), and a human subject. In comparison to the human subject, both the California sea lion and the harbor seal dark-adapted relatively quickly and were more light sensitive. These findings suggest that both of these species are well suited for vision in the moderately dim shallow-water environments in which they dive to forage. In contrast, the elephant seal reached complete dark adaptation in less than half the time taken by the other pinnipeds, and it was significantly more light sensitive. Unlike the shallower-diving species, the visual abilities of the elephant seal are commensurate with the extreme conditions experienced while deep diving. Thus, we conclude that elephant seals are sufficiently adapted to rely on vision underwater, even while diving to depths in excess of 1000 meters where bioluminescence may be the sole source of ambient light.